The present invention relates to an immunoassay system and method which are based on immunospecific target-cell lysis by hemolytic virus particles.
The following publications are referred to by the corresponding number herein:
1. Choppin, P. W., and Scheid, A., Rev. Infect. Dis., 2:40 (1980) PA0 2. Shimizu, Y. K., et al., Virology, 71:48 (1976) PA0 3. Nakanishi, M., et al., Exp. Cell Res., 142:95 (1982) PA0 4. Heath, T. D., et al., Exp. Cell Res., 149:163 (1983) PA0 5. Kundrot, C. E., et al., Proc. Nat. Acad. Sci. USA, 80:1608 (1983) PA0 6. Oku, N., et al., Virology, 116:419 (1982) PA0 7. Heath, T. D., et al., ibid. PA0 8. Tomasi, M., et al., Febs. Letts., 143:252 (1982) PA0 9. Curman, B., et al., J. Biol. Chem., 255:7820 (1980) PA0 10. Heath, T. D., et al., Biochimica et Biophysica Acta, 640:66 (1981) PA0 11. Martin, F. J., et al., Biochemistry, 20:4229 (1981) PA0 12. Martin, F. J., et al., J. Biol. Chem., 257:286 (1982) PA0 13. Huang, A., et al., J. Biol. Chem., 255:8015 (1980) PA0 14. Lapidot, Y., et al., J. Lipid Res. 8:142 (1967) PA0 15. Hosaka, Y., et al., Intervirology, 10:70 (1977) PA0 16. Homma, M., J. Virol., 9:829 (1972)
An important property of hemolytic viruses is the ability to bind specifically to and fuse with target host cells (reference 1). Under isotonic or slightly hypotonic conditions, fusion with the hose cell membrane can produce cell lysis, as evidenced, for example, by release of hemoglobin from lysed erythrocytes (reference 2). The hemolytic viruses, as exemplified by Sendai virus, contain two surface glycoproteins which have been implicated in the cell binding and fusion events: an HN glycoprotein, so named because of its apparent hemagglutinating and neuraminidase activities involved in virus binding to and cleavage of a sialic acid moiety associated with the host cell receptor; and an F glycoprotein, so named because of its apparent role in fusion of the attached virus with the host cell membrane (reference 3).
The interaction of hemagglutinating viruses with target cells have been studied in a number of surface-modified and/or artificial systems. Early studies of this type showed that the binding and lytic activity of hemolytic virus, e.g. Sendai virus, toward erythrocytes is substantially eliminated when the erythrocyte target cells are desialylated by treatment with neuraminidase (reference 4). Kundrot, et al. demonstrated that lipid vesicles (liposomes) containing cholesterol and surface-bound glycophorin (the Sendai-virus receptor on human erythrocytes) are susceptible to lysis by intact Sendai virus, but not by virus which have been inactivated by trypsin digestion (reference 5). Similarly, Oku, et al. have observed that liposomes composed of phosphatidylcholine (PC) and surface-bound glycophorin were damaged by native, but not by trypsin-treated hemagglutinating virus (reference 6). The above studies indicate that cell lysis is mediated by specific binding interaction between one or more trypsin-labile viral glycoproteins and cell-bound glycophorin.
More recent studies have examined the nature of the virus/cell binding specificity in the cell lytic reaction. In one such study, Heath, et al. have shown that Sendai virus modified to contain surface-bound anti-glycophorin can hemolyze desialylated human erythrocytes, the lytic reaction presumably being mediated by binding of virus-bound anti-glycophorin to cell-surface glycophorin (reference 7). It has also been reported that virus particles having surface-bound anti-erythrocyte antibodies are capable of lysing neuraminidase-treated red blood cells (reference 8). The latter report suggests that cell lysis can be mediated by a virus/cell binding event between virus-bound anti-erythrocyte antibodies and non-glycophorin erythrocyte surface antigen(s). However, as emphasized in the report, the "targeted virus" produced only about 1% to 10% of the hemolytic activity obtained with untreated virus and normal erythrocytes.
The present invention includes a novel immunoassay system in which hemolytic virus particles carrying non-viral, analyte-binding molecules interact specifically with foreign binding molecules anchored to target cells, to produce efficient target cell lysis. In some cases, the efficiency of cell lysis observed is substantially 100% that of normal viral-mediated cell lysis involving native viral and target cell components. The system of the invention includes hemolytic particles having surface-bound paramyxovirus HN and F glycoproteins, and surface arrays of non-viral, anti-analyte molecules. Target cells in the system are substantially devoid of surface receptors capable of binding to such HN or F proteins, and are prepared to include an encapsulated reporter which is detectable upon cell lysis. Foreign binding molecules anchored to the cells function to attach the hemolytic particles to the cell surfaces, to initiate target cell lysis, as evidenced by reporter release from the cells.
The immunoassay system may be used in two general types of reactions for the determination and/or quantitation of soluble analytes. In one reaction, referred to herein as a competitive-inhibition reaction, the foreign binding molecules include analyte-like molecules i.e., analyte or analyte analogue molecules which are attached to the cell surfaces and which are capable of competing with the analyte for binding to the particle anti-analyte molecules. A greater concentration of analyte molecules in the assay mixture therefore produces proportionately less binding of the hemolytic particles to the target cells and less released reporter. The competitive inhibition assay can be used for determination of analytes capable of one (monovalent) or more (multivalent) binding reactions. In the second type of reaction, referred to herein as a sandwich-type reaction, the foreign binding molecules are soluble analyte molecules capable of reacting cooperatively both with analyte-related molecules attached to the target cell surfaces, and with particle anti-analyte molecules, to bind the particles to the cells by analyte bridging. The soluble analyte molecules in this type of reaction are necessarily multivalent, i.e. contain at least two specific binding sites which can participate in cooperative binding to the target cell and particle surface molecules. The cell lysis immunoassay reactions, and particularly the sandwich-type reaction, have the potential for high assay sensitivity, since relatively few analyte-related binding events on the target cell surface can lead to the release or expression of a large number of reporter molecules.
The reporter which is encapsulated in the target cells may include cell-endogenous molecules, such as hemoglobin reporter molecules contained within surface-modified erythrocytes. Alternatively, the target cells may be prepared to encapsulate exogenous reporter molecules such as enzymes, chromophores, fluorogenic compounds or stable electron paramagnetic compounds. With many of the non-chromophoric reporter molecules just mentioned, released reporter can be readily detected in a suspension of target cells, affording the advantage that the complete reaction procedure--including the analyte-dependent binding reaction between the particles and cells, and measurement of released reporter form lysed target cells--can be performed in a single assay mixture, without the requirement for an intervening fractionation step. Immunoassays which can be carried out in this fashion are generally referred to as homogeneous immunoassays.
Also included in the invention is an immunoassay kit for the determination and/or quantitation of a soluble monovalent or multivalent analyte. The kit includes the hemolytic particles and target cells described above.
The invention further includes a method for the determination of a soluble analyte, including the steps of (1) providing hemolytic virus particles and lysable cells of the type described above; (2) reacting the virus particles and cells with a soluble analyte, to bind virus particles to the cells in proportion to the amount of analyte present; and (3) determining the extent of cell lysis.
One general object of the present invention is to provide a novel viral lysis immunoassay for determination of soluble monovalent and multivalent analytes.
Another object of the invention to provide a simple, homogeneous immunoassay for determination of such analytes.
Yet another object of the invention is to provide such an immunoassay which utilizes components which are easily prepared and stable on storage.
These and other objects and features of the present invention will become more fully apparent from the following detailed description of the invention.